Tissue equivalents

ABSTRACT

A method of forming a tissue equivalent is described. A length of polyester tube is threaded over a mandrel (H) and attached at either end by clips at A and D. A block (F) is then screwed into place. The polyester is then pre-soaked by injecting through Tap ( 2 ) an acidified collagen solution for approximately one hour. After a suitable period of time the excess solution is aspirated off. Following this stage, a second, alkaline solution is injected into the apparatus which contains smooth muscle cells (SMC). Thus, neutralisation of the collagen impregnated within the fabric of the tube occurs leading to spontaneous fibrillogenesis within the interstices of the cloth, eliminating the risk of delamination. The apparatus is then incubated. The collagen contracts down onto the fabric tube and the cell-impregnated gel becomes incorporated into the presoaked collagen. The pre-impregnated collagen and the collagen provided in the aqueous mixture contract down as one into a coherent whole with the SMC. The tissue equivalent is then lined with endothelial cells and the apparatus again incubated and fed at increasing intraluminal pressures applied either statically or dynamically. This is believed to cause the vessel to become preconditioned to the pressure it will work under as an implant. This allows organization of the basement membrane which in turn promotes EC attachment and theoretically prevents smooth muscle cell hyperplasia.

INTRODUCTION

This invention is concerned with the creation of tissue equivalents andmethods for their preparation. Although reference will be madehereinafter to the preparation of vascular grafts, it should beunderstood that the present invention has applications to various othertissue equivalents, for instance heart valves. The invention hasapplication to a suitable implantable or reconstructive material with anactive cellular lining, for example endothelial, whether autologous ornot.

BACKGROUND TO THE INVENTION

There are a number of clinical situations in which implants arerequired. A vascular graft may be desirable in order to replace asection of vessel damage during trauma, or to bypass vessels exhibitingocclusive diseases, for instance, for coronary artery bypass. The twomajor alternatives available for vascular grafting are the use of anautologous vessel from elsewhere in the body, or the use of biologicalor synthetic prostheses. Both alternatives have drawbacks. The formermay be surgically time consuming and availability limited. Prostheticmaterials, while surgically convenient, have a number of performancedisadvantages.

Coronary artery bypass grafting remains the most effective longer termtreatment for coronary artery disease. Since the introduction of thebypass technique, many different types of vascular grafts have beenevaluated. The most obvious source of a vascular conduit is autologousvein or artery removed or transposed from elsewhere in the body.Saphenous vein and internal mammary artery have been used. Saphenousvein conduits suffer from early thrombotic occlusion and also from latefailures so that more than 50% have occluded by 10 years. A majordrawback with the use of autologous material is that, when occlusiondoes occur, there is often insufficient autologous conduit left forre-operations. This is particularly relevant when multiple bypassoperations are required. Alternatively, saphenous vein may beunavailable, for instance, due to varicosities.

Artificial prostheses constructed from such diverse materials as wovenDacron™, Gore-Tex™ and expanded microporous polytetrafluoroethylene allsuffer to a greater or lesser extent from thrombus formation. Attentionhas therefore turned towards biological grafts of non-autologous origin.

Allograft veins and arteries from post mortem donors have been used withvariable success. However, reliance on this source is unlikely to fulfildemand. To overcome this availability problem, the use of humanumbilical veins and arteries has been considered. Unfortunately, thesehave exhibited poor mechanical performance and rapid occlusion.

Following the successful use of glutaraldehyde preserved animal valvesin valve replacement surgery, many groups turned to xenografts as apossible source of vascular replacements. Xenografts need to bechemically modified in order to decrease immunogenicity and increaseresistance to resorption. Glutaraldehyde has been the commonestcrosslinking agent used. However, its side effects include cytotoxicitywhich could inhibit endothelial re-colonisation and increased stiffeningleading to kinking and a lack of stretch and elasticity. When a lesssevere crosslinking regimen was used, using aldehyde vapour, only atemporary resistance to degradation was seen. This is likely to lead toan increased risk of aneurysm formation.

U.S. Pat. No. 4,546,500 discloses the preparation of a vessel equivalentfrom a series of layers of gel contracted from aqueous mixtures ofcollagen fibrils, a nutrient medium and smooth muscle cells orfibroblast cells. In addition, it has been suggested to include aDacron™ mesh between two layers of the multi-layer structure. Theaddition of a second layer of collagen resulted in a multi-laminatedstructure in which the mechanical strength was provided by the Dacron™alone. These blood vessel equivalents, constructed from several layersof collagen and Dacron™, have been shown to suffer from delaminationproblems. The use of fibrin as a biological glue to adhere the collagenlayers together has since been employed. It is an object of thisinvention to overcome this problem.

In addition, it is an object of this invention to improve thepreconditioning of the tissue equivalents to the pressures—e.g. bloodpressure—to which it will be subjected within the body.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention, there is provideda method of forming a tissue equivalent comprising impregnating afibrous substrate with collagen fibrils, applying to the impregnatedsubstrate an aqueous mixture of collagen fibrils, a nutrient medium anda cellular contractile agent, and incubating the system to allow a gelto form from the aqueous mixture and to contract and express aqueousmedium therefrom. By pre-impregnating the fibrous substrate withsolubilised collagen, followed by casting a single collagen gel, theproblems of delamination are avoided. Using the process of the presentinvention, collagen has been shown to infiltrate and interweave withinthe synthetic structure and to be reorganised within it an a coherentwhole. The preferred fibrous substrate is Dacron™ fabric.

The fibrous substrate may be impregnated by soaking in an acidic aqueouscollagen solution. This is preferable as it holds the collagen insolution during the impregnation. Subsequently, an alkaline aqueousmixture of collagen fibrils, a nutrient medium and a cellularcontractile agent may be applied to neutralise the acidic impregnationof the substrate and initiate collagen fibrillogenesis. The cellularcontractile agent will usually comprise smooth muscle cells.

Thrombogenicity may be prevented by covering the tissue equivalent witha monolayer of functional endothelial cells (EC). The attachment andactivity of these cells may be enhanced by their interaction with theunderlying SMC populated collagen matrix, resulting in the formation ofbasement membrane.

According to a second aspect of the present invention, there is provideda method of forming a tissue equivalent comprising forming a contractedgel from an aqueous mixture of collagen fibrils, a nutrient medium and acellular contractile agent, lining the tissue equivalent withendothelial cells, applying an aqueous fluid under pressure to the faceor faces of the tissue equivalent lined with endothelial cells andpreconditioning the tissue equivalent by incubating the system whilstincreasing the pressure applied to the said face or faces of the tissueequivalent. The endothelial cells applied to a tissue equivalent formedin accordance with the first aspect of the invention may be applied inthis way. By using hydrostatic and/or hydrodynamic preconditioning, theremodelling of the collagen fibrils can be influenced. In this way, thealignment of the resulting fibres may be of structural importance asthey tend to align in such a way as to account for the stresses imposedby the pressurised fluid.

The lining of the tissue equivalent with endothelial cells may involveexposing it to cultured endothelial cells in a cell support medium.Similarly, the pressurised may be an endothelial support medium.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will now be described by way of example withreference to the accompanying drawing of apparatus upon which a vesselequivalent may be formed.

DETAILED DESCRIPTION

The present invention, in its application to vascular grafts, isconcerned with the production of material having improved performancecharacteristics which is suitable for use in a variety of clinicalindications, including coronary bypass surgery, and which is alsosurgically convenient.

To produce a vascular graft in accordance with the presently describedexample of the invention, a tube is constructed from human vascularcells, extracellular matrix macromolecules, and a synthetic polymer suchas polyester. This fabrication process makes use of the ability ofvascular smooth muscle cells to remodel and reorganise anhydrated type Icollagen gel into a tissue-like material. By combining this “livingtissue” with a synthetic tubular framework, and subsequently lining thelumen with functional endothelium, a viable vascular graft may beformed. Ideally, for a graft to be successful, it must benon-immunogenic, non-thrombogenic and biocompatible. It must also beable to withstand intraluminal pressures in the order of 300 mmHg whilstmaintaining its structural integrity, i.e. it must not leak or formaneurysm.

By using autologous cells, problems of immunogenicity are circumvented.However, it is also possible to use allogenic cultured cells withouteliciting a graft-specific immune response. The collagen used may beextracted from rat tail tendons, in the form of an acid solubilisedsolution, or commercially available collagen powders. This collagensolution is combined with serum, culture medium and smooth muscle cells(SMC) to form the casting solution. None of these constituents will besignificantly immunogenic. Neutralisation of the casting mixture inducesfibrillogenesis of the collagen and the formation of a gel. The collagenis subsequently reorganised and structured into thicker fibres throughthe action of the SMC.

Biocompatibility refers to the synthetic element of the proposed graft.By using medical grade materials, this is not a problem. The biologicalcomponent of the composite, i.e. the collagen/SMC matrix, is the mostvulnerable to degradation. Experimental evidence indicates that thecollagen matrix becomes increasingly resistant to collagenase digestionas time in culture progresses. This indicates crosslinking of thecollagen by the cell dependent reorganisation process. Furthercrosslinking, using chemical agents may be carried out.

The cells utilised in this invention may be autologous or otherwise andare cultured using standard cell culture techniques. Additions to thestandard solutions for the gel matrix may include various proteoglycanse.g. Chrondroitin 6 Sulphate, and/or Hyaluronic acid. The culturesolutions may be varied. Accepted sterile techniques should be utilisedthroughout.

The mechanical strength of the composite graft is derived from twosources: the synthetic support; and the collagen matrix. By means of amethod of the present invention, it is possible to construct a smallcalibre vascular graft, or indeed larger grafts, with a functionalendothelium. The structure of this graft consists of a living component(collagen/SMC/EC) and a non-living synthetic element. This graft is atrue composite, both components providing the mechanical strength. Thisapproach is in contrast to known methods in which a Dacron™ sleeve isadded to the first layer of collagen after the collagen reorganisationwas completed.

The presence of living endothelial cells on the vessels internalsurface, which actively produce substances able to prevent thrombosisformation has major advantages to graft patency. The collagen/SMC matrixmimics the sub-endothelial layer of blood vessel walls, thus providing astructure for anchoring the endothelial cells to the vessel wall. Such acomposite allows the formation of a functional basement membrane. Webelieve that: the basement membrane is important not only in allowinglining regeneration after injury but also in protecting against smoothmuscle cell hyperplasia which reduces vessel patency.

EXAMPLE

The following description relates to a preferred method of creating atubular vessel equivalent. The apparatus of FIG. 1 is currently beingemployed—but many variations could be made by one of ordinary skill inthe art to the basic design without departing from the invention. Thisexample creates a craft which will not delaminate.

The dimensions of the apparatus vary depending on the diameter andlength of graft required. The apparatus should be made of a materialwhich can be sterilised without becoming opaque. The apparatus consistsof a Perspex™ chamber G sealed at its ends by blocks E & F, presentlymade from Delrin™. The central mandrel H may be of any non-toxicnon-degradable material—for very small diameters a non-absorbable suturemay be used but it must not adhere to the gel. It is a temporary supportfor the graft. The 3-way taps 1, 2, 3 and 4 are of medical grade.

Once the apparatus has been sterilised block F is unscrewed and asuitable length of polyester tube of the required diameter is threadedover mandrel H and attached at either end by clips at A & D. F is thenscrewed back into place. In this description knitted Dacron™ polyestermaterial is used, made by Vascutek Limited, Renfrewshire, Scotland.Various materials woven or knitted of implant grade—sterile andbio-compatible—may be used.

Once the Dacron™ is in place it is then pre-soaked by injecting throughTap 2 an acidified collagen solution for approximately one hour—thoughthe timing is not critical. The solution contains purified collagen at 1mg/ml of 1:1000 acetic acid solution. This; concentration may be varied.After such a period of time the excess solution is aspirated off. Thistechnique ensures impregnation of the fabric tube.

Following this stage, a second solution is injected into the apparatuswhich contains smooth muscle cells (SMC). The passage number of thecells may be varied as they may also be for the endothelial cells (EC)discussed below. The seeding concentration of the cells may also bevaried which allows faster or slower concentration time. The seedingdensity described below used 3.3×10⁴ per 5 mls of matrix solution.

The matrix solution contains 36 mls×2 Dulbecco's Modified Eagles Medium(DMEM). In all, 9 mls Pooled Human Serum, 9 mls 0.1 M NaOH, 27 mlsCollagen at 3.3 mg/ml and 9 mls of SMC were present in 1 unit of DMEM.The nutrient medium may be altered from the above and it is within theability of one of ordinary skill in the art to do so.

Solution may also be injected into the lumen by manipulating taps 1 & 4.Thus, neutralisation of the collagen impregnated within the fabric ofthe tube occurs leading to spontaneous fibrillogenesis within theinterstices of the cloth, reducing the risk of delamination. Theapparatus is then incubated at 37° C. using standard cell culturedevices. After 3-5 days depending on cell concentration for example, thecollagen contracts down onto the fabric tube and the cell-impregnatedgel becomes incorporated into the presoaked collagen. Thepre-impregnated collagen and the collagen provided in the aqueousmixture contract down as one into a coherent whole with the SMC.

Nutrient Medium, e.g. DMEM, together with human serum with or withoutfoetal calf serum, will be added to the apparatus every 3-5 days tomaintain the cells and matrix after the existing solution has beendrained off. After a period of approximately 21 days—again varying withtemperature and cell seeding density, collagen concentration etc., thematrix will have contracted sufficiently for the next stage, which isendothelial seeding.

Standard cultured endothelial cells, of variable cell lines and passagenumber,, are then added to the inside of the tube after being mixed witha standard endothelial support medium which includes heparin, human andfoetal calf serum and endothelial cell growth factors. Endothelialseeding density may vary but in this example 5×10⁴ per cm² is used. Theapparatus is then gently rolled and agitated to spread the EC's on theluminal surface for a variable but noncritical time span.

The apparatus is then again incubated and a confluent monolayer of EC'sobtained within usually one week. Standard endothelial support mediumwith its growth factor etc., will be exchanged with the existing fluidevery few days. A basement membrane is thus created within a shortperiod of time. The vessel is kept incubated for a relatively shortperiod of time, e.g. one week, and fed at increasing intraluminalpressures applied either statically or dynamically. For example aperistaltic pump may circulate the support medium in a close loop at therequired pressures. We believe that this causes the vessel to becomepreconditioned to the pressure it will work under as an implant. Thisallows organisation of the basement membrane which in turn promotes ECattachment and theoretically prevents smooth muscle cell hyperplasia.

Such a graft may then be implanted or cryopreserved using standardCyopreservation techniques. In addition, the exterior of the tissueequivalent may be sealed with biological glue.

The essentials of the two aspects of this invention are the two stagecasting of the extracellular gel into and on the Dacron™ fabric and thepreconditioning of the graft. The nature and constituents of thesolutions may readily be varied and within the standard knowledge ofcell culture techniques are not in themselves critical.

What is claimed is:
 1. A method for forming and preparing for use atissue equivalent, comprising the steps of: forming a contracted gelfrom an aqueous mixture of collagen fibrils, a nutrient medium and acellular contractile agent; lining the contracted gel with endothelialcells, thereby obtaining a tissue equivalent; applying an aqueous fluidunder pressure to at least one face of said tissue equivalent; and,preconditioning said tissue equivalent by incubation, while increasingthe pressure applied to said at least one face of said tissueequivalent, said preconditioning taking place only after completion ofsaid lining the contracted gel with endothelial cells to obtain saidtissue equivalent.
 2. The method for forming and preparing a tissueequivalent according to claim 1, wherein said step of lining thecontracted gel with endothelial cells comprises exposing the contractedgel to cultured endothelial cells in a cell support medium.
 3. Themethod for forming and preparing a tissue equivalent according to claim1, where the pressure applied is hydrostatic.
 4. The method for formingand preparing a tissue equivalent according to claim 1, wherein saidaqueous fluid flows under pressure across said at least one face of saidtissue equivalent.
 5. The method for forming and preparing a tissueequivalent according to claim 1, wherein said aqueous fluid is anendothelial support.